Spencer Hermanson

Screening for Novel LRRK2 Inhibitors Using a High-Throughput TR-FRET Cellular Assay for LRRK2 Ser935 Phosphorylation

Background Mutations in the leucine-rich repeat kinase-2 (LRRK2) have been linked to Parkinson’s disease. Recent studies show that inhibition of LRRK2 kinase activity decreased the level of phosphorylation at its own Ser910 and Ser935, indicating that these sites are prime targets for cellular readouts of LRRK2 inhibition. Methodology/Principal Findings Using Time-Resolved Förster Resonance Energy Transfer (TR-FRET) technology, we developed a high-throughput cellular assay for monitoring LRRK2 phosphorylation at Ser935. LRRK2-Green Fluorescence Protein (GFP) fusions were expressed in cells via BacMam. Phosphorylation at Ser935 in these cells is detected using a terbium labeled anti-phospho-Ser935 antibody that generates a TR-FRET signal between terbium and GFP. LRRK2 wild-type and G2019S are constitutively phosphorylated at Ser935 in cells as measured by TR-FRET. The phosphorylation level is reduced for the R1441C mutant and little could be detected for the kinase-dead mutant D1994A. The TR-FRET cellular assay was further validated using reported LRRK2 inhibitors including LRRK2-IN-1 and our results confirmed that inhibition of LRRK2 can reduce the phosphorylation level at Ser935. To demonstrate the utility of this assay for screening, we profiled a small library of 1120 compounds. Three known LRRK2 inhibitors were identified and 16 hits were followed up in the TR-FRET and a cytotoxicity assay. Interestingly, out of the top 16 hits, five are known inhibitors of IκB phosphorylation, two CHK1 and two CDC25 inhibitors. Thirteen hits were further tested in a biochemical LRRK2 kinase activity assay and Western blot analysis for their effects on the phosphorylation of Ser910, Ser935, Ser955 and Ser973. Conclusions/Significance We developed a TR-FRET cellular assay for LRRK2 Ser935 phosphorylation that can be applied to the screening for LRRK2 inhibitors. We report for the first time that several compounds such as IKK16, CHK1 inhibitors and GW441756 can inhibit LRRK2 Ser935 phosphorylation in cells and LRRK2 kinase activity in vitro.

A quantitative TR-FRET plate reader immunoassay for measuring autophagy.

Autophagy involves the isolation and targeting of unwanted cellular components to lysosomes for their digestion and reuse. Autophagic dysregulation has recently been implicated in a wide range of disease processes, yet facile methods for quantifying autophagy have been lacking in the field. Here we describe the generation of a quantitative plate reader assay for measuring the autophagic activity of cells. One of the best characterized autophagy markers is the protein LC3B, which normally resides in the cytosol (LC3B-I) but upon induction of autophagy becomes lipidated and embedded in autophagosomal membranes (LC3B-II). To quantify autophagy, we reasoned that GFP-tagged LC3B could serve as a time-resolved fluorescence resonance energy transfer (TR-FRET) acceptor upon cell lysis in the presence of terbium-labeled LC3B antibodies. Using this TR-FRET immunoassay approach, we screened a panel of LC3B antibodies and identified an antibody that exhibits strong preferential affinity toward autophagosome-associated LC3B-II and thereby facilitates specific detection of autophagic activity. The plate reader format provides both a quantitative and an objective result, thus overcoming some of the key limitations of the traditional immunoblotting and imaging approaches used to monitor autophagy. Moreover, since the assay step requires only a single addition of cell lysis buffer containing the detection antibody its simple workflow is both automation-friendly and scalable, which renders it suitable for high-throughput screening. We demonstrate how this TR-FRET immunoassay for GFP-tagged LC3B-II can be applied to quantitatively detect changes in the autophagy activity of cells, including estimating effects on autophagic flux.

Pharmacological inhibition of LRRK2 cellular phosphorylation sites provides insight into LRRK2 biology.

Mutations in LRRK2 (leucine-rich repeat kinase 2) have been linked to inherited forms of PD (Parkinsons disease). Substantial pre-clinical research and drug discovery efforts have focused on LRRK2 with the hope that small-molecule inhibitors of the enzyme may be valuable for the treatment or prevention of the onset of PD. The pathway to develop therapeutic or neuroprotective agents based on LRRK2 function (i.e. kinase activity) has been facilitated by the development of both biochemical and cell-based assays for LRRK2. LRRK2 is phosphorylated on Ser910, Ser935, Ser955 and Ser973 in the N-terminal domain of the enzyme, and these sites of phosphorylation are likely to be regulated by upstream enzymes in an LRRK2 kinase-activity-dependent manner. Knowledge of these phosphorylation sites and their regulation can be adapted to high-throughput-screening-amenable platforms. The present review describes the utilization of LRRK2 phosphorylation as indicators of enzyme inhibition, as well as how such assays can be used to deconvolute the pathways in which LRRK2 plays a role.

Development and Validation of a Cell-Based Assay for the Nuclear Receptor Retinoid-Related Orphan Receptor Gamma

The nuclear receptor retinoid-related orphan receptor gamma (RORγ) has become an attractive target for drug discovery due to its important role in the development and differentiation of Th17 cells, a subset of T cells that produce interleukin-17 and are involved in the pathogenesis of human inflammatory and autoimmune diseases. To facilitate the drug discovery efforts in this area, we have developed a cellular assay for screening for RORγ inverse agonists. We stably engineered a tetracycline-inducible Gal4 DNA-binding domain/RORγ ligand-binding domain fusion protein into an upstream activation sequence driven-beta-lactamase reporter gene cell line. Due to its constitutive activity, the induced Gal4-RORγ expression leads to increased reporter activity, which can be knocked down using RORγ ligand-binding domain-specific RNA interference oligos. Using this assay, we tested several recently reported ligands for RORγ and observed varying levels of partial inverse agonist activity at μM concentrations. Additionally, we screened a small library of biologically active compounds with this assay and demonstrated its robustness and usefulness in high-throughput screening and follow-up studies for this emerging drug target.

Abstract 3881: BacMam-enabled biochemical and cellular assays to assess inhibitors of full-length receptor tyrosine kinases

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Receptor tyrosine kinases (RTKs) are important pharmacological targets in oncology. Current biochemical assays designed to assess compound efficacy utilize truncated forms of the kinase lacking the transmembrane or extracellular domains and fail to address how these domains might affect the observed pharmacology. Purification of the full-length recombinant receptor can be both costly and difficult to scale for large screening campaigns. Additionally, because of the generic nature of most substrates used in activity assays for RTKs, small amounts of contaminating kinases can interfere with assay results. Numerous cell-based assays for pathway analysis can also serve as readouts for these targets, but they are not a direct measurement of kinase activity. To address these limitations, we have employed a BacMam-mediated gene delivery system to express full-length receptor tyrosine kinases with C-terminal green fluorescent protein (GFP) fusions in a variety of cellular backgrounds. Expression of these full-length RTKs has enabled the development of two complimentary assay platforms. First, we present a competitive displacement assay that uses a europium (Eu)-labeled anti-GFP antibody and an AlexaFluor® 647-labeled active site probe to characterize compound binding to the RTK in a lysate-based format. Here, the GFP moiety serves as an epitope tag to allow for very specific and sensitive detection of compound binding, discriminating compounds with sub-nanomolar affinity. Using the same BacMam RTK-GFP constructs, we have also developed a simple, addition-only, cell-based method to detect auto-phosphorylation of the RTK via TR-FRET between a terbium (Tb)-labeled anti-phospho-tyrosine antibody and the GFP moiety. Together, these tools can be used in a high-throughput screening format with a fluorescence-based readout to more fully characterize compound efficacy against full-length RTKs expressed in native cellular contexts. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3881. doi:1538-7445.AM2012-3881

Abstract 5006: High-throughput TR-FRET cellular assays for interrogating histone H3 methylation

Post-translational modifications such as phosphorylation, acetylation and methylation play important roles in regulating the structures and functions of histones, which in turn regulate gene expression and DNA repair and replication. Histone modifying enzymes, such as deacetylases, methyltransferases and demethylases, have been pursued as therapeutic targets for various diseases. However, detection of the activities of these enzymes in high-throughput cell-based formats has remained challenging. We have developed high-throughput LanthaScreen ® cellular assays for histone H3 site-specific modifications. These assays utilize cells expressing green fluorescence protein (GFP) tagged-histone H3 transiently delivered via BacMam and terbium labeled-anti-histone H3 modification-specific antibodies. Robust time-resolved Foerster resonance energy transfer signals were detected for H3 lysine-9 di-methylation (K9me2), lysine-4 di- (K4me2) and tri-methylation (K4me3) and lysine-27 tri-methylation (K27me3). Consistent with previous reports, hypoxic stress increased K4 methylation levels and methyltransferase G9a inhibitor UNC-0638 decreased K9me2 levels significantly with little effects on other modifications. Further validation of these assays using methyltransferases-specific RNAi oligos demonstrated that EZH2-specific RNAi reduced the level of K27me3 with little effect on the other three modifications, whereas G9a RNAi and SMYD3 RNAi reduced K9me2 and K4me2/3, respectively. The use of BacMam gene delivery system enables the measurement of these histone methylations in a variety of cell backgrounds including primary cells. In conclusion, we have developed homogenous cellular assays that can enable the high-throughput screening for modulators of histone H3 methylation at various lysine sites. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5006. doi:1538-7445.AM2012-5006

Abstract 67: High-throughput cellular assays for interrogating epigenetic histone modifications

Epigenetic histone modifications such as, methylation, acetylation, and phosphorylation play important roles in regulating the structure and functions of histones which in turn regulate essential gene expression. Dysregulation of the enzymes responsible for these modifications has been linked to cancer and many other diseases. In particular, histone deacetylases (HDACs) and histone methyl transferase (HMTs) have been a recent focus for drug discovery. However, detection of these enzymatic activities in an HTS cell-based format has remained challenging, especially since many of these enzymes may require the formation of protein complexes for relevant activity. To help overcome these challenges, we have developed and validated high-throughput compatible LanthaScreen ® cellular assays for the analysis of histone methylation, acetylation, and phoshorylation, in cell backgrounds of interest. The acetylation, methylation and phosphorylation of H3 at specific residues can be detected with terbium-labeled antibodies against the specific site and modification. The expression of GFP-H3 substrate allows for detection of antibody binding in cell lysates via TR-FRET between the terbium and GFP. Since the actual modification takes place in the intact cell, native protein complexes that may be important for enzyme function are preserved. Together, these assays enable interrogation of multiple enzymatic activities responsible for epigenetic modifications of challenging histone targets Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 67. doi:10.1158/1538-7445.AM2011-67

Abstract 4867: High-throughput LanthaScreen® cellular assays for interrogating post-translational modifications of p53 and histones

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Post-translational modifications such as phosphorylation, acetylation and ubquitination play important roles in regulating the structure and functions of histones and transcription factors such as p53, which in turn regulate essential gene expression. Dysregulation of these post-translational modifications has been linked to cancer and metabolic diseases. In particular, histone deacetylases (HDACs) have been pursued as valuable targets for various therapeutic interventions. However, detection of these enzymatic activities in a cell-based format has remained intractable using HTS-compatible technologies. To enable the drug discovery for these post-translational modification enzymes, we have developed and validated high-throughput compatible LanthaScreen® cellular assays for the analysis of histone and/or p53-specific acetylation, ubquitination and phoshorylation in cell backgrounds of interest. The cell cycle-dependent acetylation of histone H3 at Lys9 and the phosphorylation of histone H3 at Ser10 can be detected with terbium-labeled anti-Histone H3 acetyl-lys 9 and anti-phospho-Ser10 specific antibodies, respectively. In addition, poly-ubiquitination of Histone H2B can be measured with terbium-labeled anti-poly-ubiquitin antibody. We have also applied this technology and developed cellular assays for measuring DNA-damage induced phosphorylation at Ser15 and acetylation at Lys382 of p53. These assays were further validated with both small molecule inhibitor and siRNA against specific HDAC family members. Our results suggest that the deacetylation of Histone H3 Lys 9 is mediated by type I/II HDACs, whereas the deacetylation of p53 at Lys382 is mediated synergistically by both SIRT1 and type I/II HDAC activities. Together, these assays enable interrogation of multiple enzymatic activities responsible for post-translational modifications of challenging targets such as histones and p53. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4867.